CN102395353A - Composition for improving skin conditions using fetal mesenchymal stem cells from amniotic fluid - Google Patents

Composition for improving skin conditions using fetal mesenchymal stem cells from amniotic fluid Download PDF

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Publication number
CN102395353A
CN102395353A CN2010800169255A CN201080016925A CN102395353A CN 102395353 A CN102395353 A CN 102395353A CN 2010800169255 A CN2010800169255 A CN 2010800169255A CN 201080016925 A CN201080016925 A CN 201080016925A CN 102395353 A CN102395353 A CN 102395353A
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cell
amniotic fluid
stem cell
culture medium
fetus
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Inventor
刘承权
尹炳善
文哉喜
全恩暻
金钟建
郑惠年
李重翰
朴乙纯
孟利朔
金俊成
李章浩
李黄熙
李宗远
曹敬植
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Stemmedience Co Ltd
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Stemmedience Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Abstract

The present invention relates to a medium for fetal mesenchymal stem cells from amniotic fluid. More specifically, the present invention relates to a composition for improving skin conditions, which comprises the medium for fetal mesenchymal stem cells from amniotic fluid as an active ingredient, wherein the skin conditions include whitening, wrinkles, skin damages caused by UV rays and skin lifting. In addition, the present invention relates to a preparation method of the composition, which comprises: incubating fetal mesenchymal stem cells from amniotic fluid; and collecting the medium.

Description

Use the compositions of improving skin from the fetus interstital stem cell of amniotic fluid
Background of invention
1. technical field
The present invention relates to the culture medium of fetus source interstital stem cell in the amniotic fluid.More specifically; The present invention relates to be used to improve the compositions of skin; It comprises the culture medium of the fetus source interstital stem cell in the amniotic fluid as effective ingredient, wherein skin comprise albefaction, wrinkling, draw by UV line or skin and to propose the skin lesion that (drawing skin, skin lifting) causes.In addition, the present invention relates to be used to prepare the method for said composition, comprise step: cultivate the fetus source interstital stem cell in the amniotic fluid; And collect this culture medium.
2. description of related art
Stem cell has through appropriate environments and is divided into the ability of various cells with stimulating, and also has the ability of self-reproduction.Three types stem cell is arranged: isolating embryonic stem cell from body early embryo (ES cell), isolating embryonic genital cell (EG cell) and from the primordial germ cell of embryo stage from adult's isolating multipotency adult CFU-GM (MAPC cell) the bone marrow.Stem cell has the potentiality of the cell that develops into have unique phenomenon (unique phenomena) and specific function, therefore they is proposed as the attractive cell source of the various organs that are used to regenerate.
So far, known adult stem has the ability that is divided into various cells.Adult stem is that (Science 276,71-74,1997 from bone marrow; Science 284,143-147,1999; Science 287,1442-1446,2000), skeletal muscle (Proc.Natl Acad.Sci.USA 96,14482-14486,1999; Nature 401,390-394,1999) and fatty tissue (Tissue Eng 7,211-228,2001; J.Cell.Physiol.206,229-237,2006) in isolating, and in them each can be divided into similar cell lineage.
The bone marrow interstital stem cell is the adult stem that has used for a long time, and their effectiveness also is proved.In addition, nearest research has reported that isolated cells also has similar characteristic to the bone marrow interstital stem cell from fatty tissue or its hetero-organization.Yet, be difficult to separate and a large amount of interstital stem cell of purification, therefore press for other optional sources.
Therefore, the inventor mainly pays close attention to and can easily be separated and baby or its mother are not caused the amniotic fluid of any danger.
After germ cell is implanted to Uterus wall several days, the embryo is filled the amniotic sac of amniotic fluid and surrounds.Because amniotic fluid comprises in a large number by the excretory material of fetus, can in amniotic fluid, check chromosomal abnormality or the bacterial infection of fetus.In addition, amniotic fluid allows fetal movements easier, and the protection fetus does not receive the influence of any external impact and stimulation, prevents bacterial infection, and helps to regulate fetus body temperature.
In utero, can obtain the information of relevant foetus health through the inspection amniotic fluid.At this moment, can collect amniotic fluid at whole phenolics, and mother not caused any danger.Cell used in the inspection abandons after inspection, and under patient's allowance, can be used for studying purpose.Therefore, because can easily obtain a large amount of cells, the amniotic fluid derived stem cell has advantage with respect to other adult stems.At present, there is not to add the report whether culture medium that obtains through fetus source interstital stem cell one period scheduled time of cultivating in the amniotic fluid influences fibroblastic growth.
The inventor fetus from amniotic fluid has separated interstital stem cell and has studied their characteristic.In addition, they after deliberation the component that exists in the conditioned medium of the fetus source interstital stem cell preparation in using amniotic fluid, and proved the effect of conditioned medium in fibroblast.In addition, the inventor is through mice in vivo test and clinical trial, proved that the conditioned medium compositions to skin elasticity and regenerated influence, accomplished the present invention thus.
Summary of the invention
An object of the present invention is to provide the compositions that is used to improve skin, it comprises the culture medium of the fetus source interstital stem cell in the amniotic fluid as effective ingredient.
Another object of the present invention provides the method that is used to prepare said composition, may further comprise the steps: cultivate the fetus source interstital stem cell in the amniotic fluid; And collect this culture medium.
Description of drawings
Fig. 1 has shown the heterogeneous population (left side) of fetus source cell system in the amniotic fluid and homogeneity enrichment (homogeneous the enrichment) (right side: 4 generations) of interstital stem cell behind 2 to 3 successive transfer culture;
Fig. 2 shown will the fetus source cell carries out karyotyping from the isolating amniotic fluid of mother the result, wherein this cell of XY sex chromosome explanation comes from the fetus in the amniotic fluid, rather than mother;
Fig. 3 has shown the result of the immunophenotype analysis of using flow cytometry, and wherein the fetus source cell (B) in the amniotic fluid has and the similar characteristic of people's bone marrow interstital stem cell (A);
Fig. 4 has shown that lipogenesis differentiation (A), Osteoblast Differentiation (B) and the cartilage of fetus source cell forms differentiation (C) in the amniotic fluid, explains that this cell has and the similar differentiation capability of multipotency bone marrow interstital stem cell;
Fig. 5 (A) has shown through using fetus source interstital stem cell and DMEM/F12-in the amniotic fluid of establishing not to contain blood serum medium preparation condition culture medium, has wherein examined under a microscope 5 * 10 4Individual (a), 1 * 10 5Individual (b), 2.5 * 10 5Individual (c) and 5 * 10 5Individual (d) cell; Fig. 5 (B) has shown at the every cm of inoculation 2Each cell counting after the result that measures of CFU;
Fig. 6 to 8 has shown the effect of raised growth factor pair fibroblastic growth, and said somatomedin is present in the conditioned medium that the fetus source interstital stem cell in the amniotic fluid of use establishing obtained;
Fig. 9 has shown the result who is used for analyzing proteinic TPPA in the conditioned medium that the fetus source interstital stem cell that uses the amniotic fluid of confirming obtained; Explanation has produced a large amount of somatomedin in culture medium ((A) DMEM/F12-does not contain the culture medium of serum, and (B) DMEM/F12-does not contain the conditioned medium of serum);
Figure 10 to 11 has shown the conditioned medium that uses the fetus source interstital stem cell in the amniotic fluid to be obtained, and in SF, has the wound healing effect;
Figure 12 has shown the result of in vivo test, explains that the conditioned medium that uses the fetus source interstital stem cell in the amniotic fluid to be obtained has regeneration in injured mouse skin; And
Figure 13 has shown the result of clinical research, explains that the conditioned medium that uses the fetus source interstital stem cell in the amniotic fluid to be obtained has regeneration.
The specific embodiment
In order to realize above purpose; An embodiment of the invention relate to the compositions that is used to improve skin; It comprises the culture medium of the fetus source interstital stem cell in the amniotic fluid as effective ingredient, wherein said skin comprise albefaction, wrinkling, draw by UV line or skin and to propose the skin lesion that causes.
As used at this; Term " interstital stem cell (MSC) " refers to the cell that is divided into cartilage, skeleton, fat, bone marrow matrix, muscle, nerve or analog; It mainly is present in adult's bone marrow; But, and can therefrom obtain also at Cord blood, come to light in blood, its hetero-organization or the analog on every side.With regard to the object of the invention, interstital stem cell of the present invention is meant the fetus source interstital stem cell in the amniotic fluid.
Be comprised in pregnant woman's the amniotic fluid by the excretory various chemicals of fetus, they can produce intravital most of cell, and are collected easily.In addition, the heterogeneous population of cell exists in amniotic fluid, and the inventor has identified the existence of the homogeneous population (homogeneous population) of the interstital stem cell with fibroblast appearance form, and said fibroblast appearance form is the characteristic of interstital stem cell.
The invention is characterized in: the cell in the amniotic fluid comes from fetus.
In preferred implementation of the present invention, carry out karyotyping to confirm whether the cell in the amniotic fluid comes from fetus.In amniotic fluid, also there be some mothers' cell and fetal cell.In the present invention, it is male to detect to carry out chromosome analysis, explains that the cell in the amniotic fluid comes from fetus (Fig. 2).
Preferably, compositions of the present invention comprises adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), VEGF (VEGF), interleukin-1 ' beta ' (IL-1 β), interleukin 1 receptor α (IL-1R α), IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-12 and IL-15 in Apo-1/Fas, epidermal growth factor (EGF), IP-10, leptin, MIP4, MMP3, Rantes, interferon gamma (IFN γ), people's transforming growth factor (TGF-β), tumor necrosis factor (TNF α), Tumor Necrosis Factor Receptors I (TNFRI), Tumor Necrosis Factor Receptors II (TNFRII), the cell.
In addition; By the fetus source interstital stem cell in the amniotic fluid of method preparation of the present invention; It is characterized in that: show (a) immunological characteristic, (b) grow and be attached to the cell culture flat board, and show fusiform form CD13, CD29 and CD44 total positives; It is typical fibroblast form, and (c) has the ability that is divided into the mesoblastema pedigree.
As used at this, term " differentiation " refers to phenomenon like this, wherein in division, propagation and growing period, its structure or the function specialization of cell.Pluripotent mesenchymal stem cell produces the CFU-GM that is divided into committed cell's pedigree (for example mesoblastema); And can further be divided into the CFU-GM (for example osteoblast etc.) of other types, it is created on the cell type (for example adipose cell, osteoblast, chondrocyte etc.) that has the differentiation of end eventually of specific function in the particular tissues (for example bone etc.) again.In a preferred embodiment, the fetus source interstital stem cell in the amniotic fluid of the present invention has the ability that is divided into adipose cell, osteoblast and chondrocyte.
In instantiation of the present invention; The culture medium that is used to be divided into adipose cell can comprise high glucose DMEM, 1mM dexamethasone, 0.5mM 3-isobutyl group-1-methyl-caffeine (3-isobutyl group-1-methyl-xanthine), 10ng/ml insulin, 100mM indomethacin and 10%FBS.
In instantiation of the present invention, be used to be divided into osteoblastic culture medium and can comprise high glucose DMEM, 100mM dexamethasone, 10mM β phosphoglycerol, 0.2mM vitamin C and 10%FBS.
In instantiation of the present invention; The culture medium that is used for differentiating cartilage-forming cell can comprise high glucose DMEM, 0.1M dexamethasone, 50g/ml AsA, 100g/ml Sodium Pyruvate, 40g/ml proline, 10ng/ml TGF-1 and 50mg/ml ITS premix [6.25g/ml insulin, 6.25g/ml transferrins, 6.25g/ml Monohydrated selenium dioxide, 1.25mg/ml BSA and 5.35mg/ml linoleic acid].
In the present invention, in culture medium, cultivate the fetus source interstital stem cell in the amniotic fluid, for each differentiation culture 7 to 28 days.By this method, whether the fetus source cell in the provable amniotic fluid has the characteristic (Fig. 4) of interstital stem cell.Therefore, in the present invention, the fetus source cell in the amniotic fluid is the cell with interstital stem cell characteristic, and can be used as the source of other cells and bone marrow.
Compositions display of the present invention improve the effect, particularly albefaction of skin, wrinkling, draw by UV line, skin regeneration or skin and to propose the skin lesion that causes.
In instantiation of the present invention, in the fibroblast of skin source, generate artificial wound, use this cell of compositions-treated of the present invention subsequently.Showed cell migration as a result improves, and the wound healing Expression of Related Genes quantitatively increases, and fabulous wound healing effect (Figure 10 to 11) has been described.
In addition, on mouse skin, generate artificial wound, use compositions of the present invention to wound subsequently.The result shows that wound site reduces gradually, and fabulous wound closure and skin regeneration effect (Figure 12) have been described.
In addition, in the clinical research of using the present composition, after little therapy (microtheraph) of the present composition; When with the microscopic needle chafe, find that skin elasticity improves, it is brighter that the colour of skin becomes; Small wrinkle reduces, and skin pore minimizes.When observing for 6 to 7 weeks in an identical manner; Observe the improvement and the wound regeneration fast of the erythema of acne scars, compositions display of the present invention is described fabulous albefaction, wrinkling, skin lesion, skin regeneration and the skin of improving draw the effect of proposing (Figure 12).
According to preferred implementation of the present invention, compositions of the present invention is a cosmetic composition.
The composition that is included in the cosmetic composition of the present invention is an effective ingredient, and the culture medium of fetus source interstital stem cell, it also comprises the composition that in cosmetic composition, uses usually in amniotic fluid.Such composition comprises for example conventional adjuvant, like antioxidant, stabilizing agent, solubilizing agent, vitamin, pigment and spice and carrier.In addition, this cosmetic composition can further comprise the skin absorbs reinforcing agent, should effect so that promote.
Cosmetic composition of the present invention can be prepared as any preparation for preparing usually in the art.This cosmetic composition can be formulated as: for example; Solution, suspension, Emulsion, paste, gel, cream, lotion, powder, soap, the cleaning agent that contains surfactant, oil, powdery foundation cream, Emulsion foundation cream, wax foundation cream, spray or analog, but be not limited thereto.More specifically, this cosmetic composition can be prepared as preparation, for example softening cosmetic water, nutrition cosmetic water, nutrition cream, massage cream, quintessence oil, eye cream, cleaning cream, cleaning foam, clean water, facial film (pack), spray or powder.If preparation of the present invention is paste, cream or gel, then animal oil, vegetable oil, wax, paraffin, starch, traganth (traganth), cellulose derivative, Polyethylene Glycol, siloxanes, bentonite, Silicon stone, Talcum, zinc oxide or analog can be used as carrier components.
If preparation of the present invention is powder or spray; Lactose, Talcum, Silicon stone, aluminium hydroxide, calcium silicates or polyamide powder can be used as carrier components so; Especially, if said preparation is a spray, can comprise propellant for example chlorofluorocarbon, propane/butane or dimethyl ether.If preparation of the present invention is solution or Emulsion; Solvent, solubilizing agent or emulsifying agent can be used as carrier components so; Its example comprises water, ethanol, boundary's propanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil (butylglycol oil), glycerin fatty family ester, Polyethylene Glycol or sorbitan fatty acid ester.If preparation of the present invention is a suspension; For example water, ethanol and propylene glycol of liquid diluent so, suspending agent for example ethoxylation isooctadecanol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitol acid anhydride ester, microcrystalline Cellulose, aluminium hydroxide, bentonite, agar, traganth or analog can be used as carrier components partially.If preparation of the present invention is the cleaning agent that contains surfactant, aliphatic alcohol sulfate, fatty alcohol ether sulphate, sulfosuccinic acid monoesters, isethionate, imidazoline
Figure BPA00001446957500061
salt derivative, methyl tauride (methyltaurate), sarcosinate, fatty acid amide ether sulfate, alkyl amido betaine, aliphatic alcohol, fatty glyceride, fatty diglycollic amide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester or analog can be used as carrier components so.
In addition, compositions of the present invention can be prepared as pharmaceutical composition.
If preparation of compositions of the present invention is a pharmaceutical composition, pharmaceutical composition so of the present invention comprises pharmaceutically acceptable carrier.Be included in the pharmaceutical composition of the present invention pharmaceutically acceptable carrier for when preparation normally used those; Comprise lactose, glucose, sucrose, sorbitol, mannitol, starch, Radix Acaciae senegalis, calcium phosphate, alginate, gelatin, calcium silicates, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate and propyl hydroxy benzoate, Talcum, magnesium stearate and mineral oil, but be not limited thereto.Except above composition, pharmaceutical composition of the present invention also can further comprise lubricant, wetting agent, sweetener, fumet, emulsifying agent, suspending agent, antiseptic or analog.Suitable pharmaceutically acceptable carrier and preparation are at Remington ' s Pharmaceutical Sciences (19 ThEd., describe 1995).
Pharmaceutical composition of the present invention can be applied to mammal for example rat, mice, domestic animal and people through number of ways; The for example oral and parenteral route of said approach; For example mouthful, injection in (intraepidural) or the cerebrovascular in rectum or intravenous, intramuscular, subcutaneous, the dura mater; Percutaneous approach in the preferred parenteral route, more preferably topical application.
Can confirm the appropriate dose of pharmaceutical composition of the present invention according to multiple factor: for example compound method (formulation method), mode of administration, patient age, body weight, sex and health status, diet, administration time, route of administration, excretion rate and drug susceptibility.For oral administration,, can once or several times use pharmaceutical composition of the present invention with the daily dose of 0.1-100mg/kg to the adult.In addition, for topical,, can preferably use pharmaceutical composition of the present invention 1 month one to five time or the longer time with 1.0 to 3.0ml daily dose to the adult.Yet scope of the present invention also is not intended to by dose limitation.
Use pharmaceutically acceptable carrier and/or excipient, according to method known to those skilled in the art, pharmaceutical composition of the present invention can be formulated into unit dosage forms or multi-dose container.This pharmaceutical composition can be formulated into any appropriate formulation, comprises oral formulations for example powder, granule, tablet, capsule, suspension, Emulsion, syrup and aerosol, local topical formulation example such as ointment and cream, suppository or the sterile solution that is used to inject.This pharmaceutical preparation can further comprise dispersant or stabilizing agent.
Other embodiments of the present invention relate to the method that is used to prepare said composition, may further comprise the steps: cultivate the fetus source interstital stem cell in the amniotic fluid; And collection culture medium.
More preferably, the present invention relates to be used to prepare the said composition method, may further comprise the steps:
(a) the fetus source cell of separation from the amniotic fluid that the pregnant woman obtains; (b) this cell of successive transfer culture in the culture medium that is supplemented with FBS and bFGF is so that obtain the fetus source interstital stem cell in the amniotic fluid; (c) in the culture medium that does not contain serum, cultivate fetus source interstital stem cell 1 to 10 day in the amniotic fluid that is obtained, so that the preparation condition culture medium; And (d) collect this conditioned medium.
In step (a), begin up to childbirth from pregnancy, all can collect amniotic fluid, and mother not caused any danger.In utero, can obtain information through amniocentesis about foetus health, employed cell abandons after inspection in the inspection.At this moment, under patient's allowance, this cell can be used for studying purpose.Therefore, can easily obtain a large amount of cells.Will be centrifugal from the amniotic fluid that mother obtains, to separate the fetus source cell in the amniotic fluid.
In step (b), successive transfer culture is isolated cells from amniotic fluid, so that obtain the fetus source interstital stem cell in the amniotic fluid.The culture medium of in successive transfer culture, using is cell culture minimal medium (CCMM) preferably, and it comprises carbon source, nitrogenous source and trace element usually.The example of cell culture minimal medium comprises DMEM (Dulbecco modification Eagle culture medium), MEM (MEM), BME (Eagle basal medium), RPMI1640, F-10, F-12, α MEM (α MEM), GMEM (Glasgow MEM) and IMEM (Iscove modification Dulbecco culture medium), but is not limited thereto.In addition, this culture medium can comprise antibiotic, for example penicillin, streptomycin and gentamycin.
In the present invention; Fetus source interstital stem cell in the amniotic fluid can obtain through in the basal medium that is supplemented with FBS and bFGF, cultivating from the amniotic fluid isolated cells, preferably obtains through cultured cell in being supplemented with the LG DMEM culture medium that contains 10%FBS of 4ng/ml bFGF.In preferred embodiment of the present invention, LG DMEM culture medium can further comprise 10%FBS, 1%L-glutamine and 1% penicillin-streptomycin, and 4ng/ml bFGF solution.
In step (c), in the culture medium that does not contain serum, cultivate the fetus source interstital stem cell 1 to 10 day in the amniotic fluid that is obtained, with the preparation condition culture medium.
As used at this; Term " conditioned medium " refers to the culture medium through the following steps preparation: when suspension cultured cells reaches exponential phase; Remove somatoblast through centrifugal or filtration, and only collect culture medium, and mix with culture matrix (culture substrate).This is to use the method that is secreted into the UGF the culture medium from somatoblast, is generally used for low-density bed board cell or is used for protoplast cultivating.
For the object of the invention; Conditioned medium compositions of the present invention refers to and comprises through from the culture medium of fetus source interstital stem cell, shifting out the compositions of the solution that fetus source interstital stem cell prepares; And refer to and comprise the big quantity of material compositions of somatomedin for example that comes from fetus source interstital stem cell, and preferably include adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), VEGF (VEGF), interleukin-1 ' beta ' (IL-1 β), interleukin 1 receptor α (IL-1R α), IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-12 and IL-15 in Apo-1/Fas, epidermal growth factor (EGF), IP-10 (interferon gamma induced protein 10), leptin, MIP4, MMP3, Rantes, interferon gamma (IFN γ), people's transforming growth factor (TGF-β), tumor necrosis factor (TNF α), Tumor Necrosis Factor Receptors I (TNFRI), Tumor Necrosis Factor Receptors II (TNFRII), the cell.
In the present invention; In order to obtain the conditioned medium of fetus source interstital stem cell; The interstital stem cell that from amniotic fluid, separates and obtain is preferably cultivated in the culture medium that does not contain serum, and this culture medium comprises Ham ' the s F-12 nutritional blend that contains aminoacid or its analog and vitamin or its analog.According to the present invention, the culture medium that does not contain serum that comprises Ham ' s F-12 nutritional blend is not based on there being the for example phenol red DMEM of pH indicator, and with about 1: the ratio of 0.5-2 is to wherein adding Ham ' s F-12 nutritional blend.About this point, can add for example L-glutaminate, energy metabolism thing Sodium Pyruvate and carbon source sodium bicarbonate for example for example of chemotrophy thing.In this mixture, help to keep cell growth cell and in the successive transfer culture after relating to the initial-stage culture that is increased in interstital stem cell with stable state safety and multiple inorganic substances and the aminoacid kept, can stimulate by vitamin nutrient and other factors of the excretory somatomedin higher yield of fetus source interstital stem cell and mix each other with given ratio.
In instantiation of the present invention, isolating fetus source cell the amniotic fluid that obtains from mother carries out successive transfer culture in the culture medium that is supplemented with FBS and bFGF, so that obtain the fetus source interstital stem cell in the amniotic fluid.Subsequently, the amniocyte of varying number does not contain in the culture medium of serum at DMEM/F-12 to be cultivated 3 days, centrifugal with filter the culture medium that is obtained so that preparation condition culture medium (Fig. 5).
In step (d), the collection condition culture medium is so that prepare the compositions that is used to improve skin of the present invention.Can known by one of skill in the art method carry out the collection of conditioned medium, for example through centrifugal or filtration.
Hereinafter, the present invention is described the reference implementation example in further detail.Yet these embodiment only are for illustrative purposes, and the present invention is not intended to by these embodiment restrictions.
Embodiment 1: cultivate the fetus source cell system from the amniotic fluid that the pregnant woman obtains, and under condition of culture, carry out the morphology evaluation of homogeneity interstital stem cell
The amniotic fluid that obtains from the pregnant woman, a lot of floating particles are arranged.In order to separate them, be placed in the T shape flask amniotic fluid and at 37 ℃ of following incubations.Second day, except the cell attached to the bottom, all cells all were removed.Use trypsin that the cell attached to the bottom is separated with the bottom, collect through centrifugal subsequently.Subsequently, with cell suspension in the basal medium of the LG DMEM that comprises 10%FBS, 1%L-glutamine and 1% penicillin-streptomycin and 4ng/ml bFGF, and with the cell inoculation that suspends in 100mm cell culture flat board.After 12-24 hour, replace this culture medium with fresh culture.Find that the fetus source cell system in the amniotic fluid shows heterogeneous population (Figure 1A: a-d), and behind two to three successive transfer culture, only have fibroblast appearance form (Figure 1A: e).When cell has the form of Fig. 1, changed culture medium once in every 2-3 days, when cell reaches 80% to 90% when converging, carry out successive transfer culture.
Embodiment 2: identify the fetus source cell in the amniotic fluid through karyotyping
In order to check that whether cell comes from the fetus in the amniotic fluid, carries out karyotyping.
Caryogram has shown quantity, size and the shape of each chromosome type, carries out chromosome analysis, to detect sudden change and definite sex of foetus.In order to carry out karyotyping, cell division was stopped in mid-term with Colchiceinamidum 1-2 hour, and show band dyeing carrying out chromosome analysis through G.The karyotyping result shows that the fetus source cell in the amniotic fluid has normal dyeing body and XY sex chromosome, explains that cell comes from fetus, rather than mother (Fig. 2).
Embodiment 3: fetus source cell in the analysis amniotic fluid and the similarity between the interstital stem cell
Checked whether the fetus source cell in the amniotic fluid has the characteristic of pluripotent mesenchymal stem cell.
At first, when carrying out the immunophenotype analysis with flow cytometry, compare, observe the expression (Fig. 3) of CD13, CD29 and CD44 in the fetus source cell in amniotic fluid with people's bone marrow interstital stem cell.Although their expression is specific for interstital stem cell, each cell all can show different characteristic.Therefore, checked the ability that is divided into osteoblast, adipose cell and chondrocyte, this is the representative characteristic of interstital stem cell.
Fetus source cell in the amniotic fluid breaks up as people's bone marrow interstital stem cell; Expression through osteopontin and osteocalcin has subsequently proved Osteoblast Differentiation; Expression through lipoprotein lipase (LPL), fatty acid binding protein 2 (aP2) and Pexoxisome proliferator activated receptor γ (PPAR γ); Proved the lipogenesis differentiation; Expression through II Collagen Type VI, type i collagen and aggrecan---through reverse transcriptional PCR (RT-PCR), proved that cartilage forms differentiation.Every kind of differentiation all uses specific colouring method to identify: the calcification during the Osteoblast Differentiation is identified with alizarin red S dyeing; The drop of lipogenesis between the idiophase gathers with oil red O stain to be identified, identifies (Fig. 4) in the cartilage matrix that cartilage formed between the idiophase with alcian blue dyeing.Therefore, find that the fetus source cell in the amniotic fluid of the present invention has the characteristic similar with the bone marrow interstital stem cell.
Embodiment 4: preparation is from the conditioned medium of the fetus source interstital stem cell in the amniotic fluid
Establish the preparation condition of conditioned medium according to the cell counting of the fetus source interstital stem cell in the amniotic fluid.
At first, through trypsin treatment, the interstital stem cell of establishing is separated from the dull and stereotyped bottom of 100mm cell culture.Institute's isolated cells is collected and is placed in the 15ml pipe, and is centrifugal subsequently.The cell suspension of collecting is mixed in the culture medium of 5-10ml and fully.The floating cell of 20 μ l in the take-off pipe, and calculate cell number with hematimeter.Subsequently, in comprising the 100mm cell culture flat board of the LG DMEM that is supplemented with 10%FBS, 1%L-glutamine and 1% penicillin-streptomycin and 4ng/ml bFGF, inoculate 5 * 10 4, 1 * 10 5, 2.5 * 10 5With 5 * 10 5Individual cell.After 12 hours, the conditioned medium that does not contain serum with DMEM/F12 replaces this culture medium, and cultured cell 72 hours.After 72 hours, each Cytometric culture medium is transferred in the pipe, and is centrifugal subsequently.Use 0.20 syringe filter to filter, with the preparation condition culture medium.DMEM/F12 do not contain cultivate 3 days in the culture medium of serum after, the form of cell is shown in Fig. 5 A after obtaining CM.Every cm 2Cell counting be divided into four groups, and inoculating cell is measured to carry out CFU.The result is presented at 100/cm 2Observe peak, and according to every cm 2Cell counting observe different patterns (Fig. 5 B).
Embodiment 5: the effect of testing in vitro conditioned medium
Counted 5 * 10 in the amniotic fluid 4, 1 * 10 5, 2.5 * 10 5With 5 * 10 5Individual fetus source interstital stem cell, and in them each all do not contain in the culture medium of serum at DMEM/F12 and cultivated 3 days, with the preparation condition culture medium.In order to check their influences, carried out following experiment to fibroblastic growth.In CM, cultivate after 3 days, compared cellular morphology and growth.At CM (2.5 * 10 5Individual) in observe the highest cell growth (Fig. 6).In order to prove this result, after handling skin source fibroblast, with PI dyeing identification of cell growth cycle with CM.As a result, at CM (2.5 * 10 5Individual) in, in S stage (propagation), observe 13.83% the highest cell growth, this identical with previous experiment (Fig. 7).In cell growth cycle, checked the relevant gene expression of S stage through PCR in real time.Find that this gene expression increases in the cell of CM processed group.Especially, at CM (2.5 * 10 5Individual) in observe high expressed, the CM that this expression comes from 250,000 amniocyte has increased cell growth (Fig. 8) greatly.On the basis of these experimental results, checked the increase that cell growth associated protein matter is expressed, to establish optimal culture conditions.
Embodiment 6: identify the conditioned medium composition in the fetus source interstital stem cell in the amniotic fluid
In order to analyze the composition that uses the conditioned medium that the fetus source interstital stem cell in the amniotic fluid obtained, checked the quantitative variation of expressing protein through TPPA., DMEM/F12 do not observe somatomedin and protein expression (Fig. 9 A) in not containing the culture medium (not being conditioned medium) of serum.Analyzed the composition that DMEM/F12 does not contain the conditioned medium of serum.As a result, find 27 kinds of somatomedin in whole 36 kinds of cell growth factor that relate to propagation and protein, for example TGF β, VEGF, EGF, TNF α, IL8, IL6 and MMP3 are included in (Fig. 9 B) in the conditioned medium.
Embodiment 7: the wound healing effect of conditioned medium in SF
Use the conditioned medium of amniotic fluid derived stem cell preparation whether to show the wound healing effect, on the fibroblast of skin source, to generate artificial wound, to handle this cell with conditioned medium subsequently in order to check.In the time of 12 hours, in the cell that conditioned medium is handled, observe higher migration, and at CM (2.5 * 10 5Individual) in much higher.About this point, compared relative migration speed.At conditioned medium (2.5 * 10 5Individual) in observe peak, explain that the conditioned medium that uses the amniotic fluid derived stem cell to obtain has fabulous wound healing effect (Figure 10 to 11).During the measurement of migration rate, checked the gene expression that relates to wound healing through PCR in real time.The culture medium (not being conditioned medium) that does not contain serum with DMEM/F12 is compared, at conditioned medium (2.5 * 10 5Individual) in observe the expression of maximum quantity, explain that the conditioned medium that comes from 250,000 amniocyte has shown the highest wound healing effect (Figure 11).
Embodiment 8: the skin regeneration effect of conditioned medium in the mice body
Whether the conditioned medium of having checked the fetus source interstital stem cell in the amniotic fluid shows the skin regeneration effect to the mice of skin trauma.Mouse skin is through perforation 29-30mm 2Injured by manual work, use common DMEM/F12 not contain the culture medium of serum subsequently and conditioned medium that DMEM/F12 does not contain serum is handled, observe wound closure.Up to 8 days, in the wound site that the culture medium that does not contain serum with common DMEM/F12 is handled, do not observe skin regeneration, but find that the wound site that does not contain the conditioned medium processing of serum with DMEM/F12 reduces (Figure 12 A) gradually.The measured value of wound closure illustrates (Figure 12 B) in the drawings.
Embodiment 9: through the effect of clinical research inspection conditioned medium
The conditioned medium of the fetus source interstital stem cell in the amniotic fluid is used for clinical research, to check its skin regeneration effect.After little therapy of conditioned medium, use the microscopic needle chafe, weekly.After 8 weeks, to observe skin elasticity and improve, it is brighter that the colour of skin becomes, and small wrinkle reduces, and skin pore minimizes (Figure 13 A).When observing 6-7 during week in an identical manner, observe the improvement and the wound regeneration (Figure 13 B) fast of the erythema of acne scars.
Effect of the present invention
In the present invention, cultivate the cell come from fetus in the amniotic fluid, obtaining pluripotent mesenchymal stem cell, and therefore except bone marrow, another interstital stem cell source is provided.In other words; The present invention proposes the cell that comes from fetus in the amniotic fluid probability as pluripotent mesenchymal stem cell; And the fetus source interstital stem cell preparation condition culture medium in the use amniotic fluid is provided for improving the fabulous compositions of cell growth and skin regeneration thus.

Claims (8)

1. compositions that is used to improve skin, it comprises the culture medium of the fetus source interstital stem cell in the amniotic fluid as effective ingredient, wherein said skin be albefaction, wrinkling, draw by UV line or skin and to propose the skin lesion that causes.
2. compositions according to claim 1, the fetus source interstital stem cell in the wherein said amniotic fluid has following characteristic:
(a) demonstration is to the immunological characteristic of CD13, CD29 and CD44 total positives;
(b) grow and be attached on the cell culture flat board, and show fusiform form; And
(c) has the ability that is divided into the mesoblastema pedigree.
3. compositions according to claim 1, the culture medium of the fetus source interstital stem cell in the wherein said amniotic fluid comprise adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), VEGF (VEGF), interleukin-1 ' beta ' (IL-1 β), interleukin 1 receptor α (IL-1R α), IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-12 and IL-15 in Apo-1/Fas, epidermal growth factor (EGF), IP-10 (interferon gamma induced protein 10), leptin, MIP4, MMP3, Rantes, interferon gamma (IFN γ), people's transforming growth factor (TGF-β), tumor necrosis factor (TNF α), Tumor Necrosis Factor Receptors I (TNFR I), Tumor Necrosis Factor Receptors II (TNFR II), the cell.
4. compositions according to claim 1, wherein said compositions are cosmetic composition.
5. compositions according to claim 4, wherein said compositions have the preparation that is selected from solution, suspension, Emulsion, paste, gel, cream, lotion, powder, soap, the cleaning agent that contains surfactant, oil, powdery foundation cream, Emulsion foundation cream, wax foundation cream and spray.
6. compositions according to claim 1, wherein said compositions are pharmaceutical composition.
7. one kind is used to prepare the described method for compositions of claim 1, may further comprise the steps: cultivate the fetus source interstital stem cell in the said amniotic fluid; And collect said culture medium.
8. one kind is used to prepare the described method for compositions of claim 1, comprising:
(a) the fetus source cell of separation from the amniotic fluid that the pregnant woman obtains;
(b) the said cell of successive transfer culture in the culture medium that is supplemented with FBS and bFGF is so that obtain the fetus source interstital stem cell in the said amniotic fluid;
(c) in the culture medium that does not contain serum, cultivate fetus source interstital stem cell 1 to 10 day in the amniotic fluid that is obtained, so that the preparation condition culture medium; And
(d) collect said conditioned medium.
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